Method of Treating or Inhibiting Malaria

ABSTRACT

Disclosed are derivative compounds of ELQ-300 that include an ester at position 4. These compounds have enhanced properties relative to ELQ-300. Also disclosed are pharmaceutical compositions comprising the compounds and methods of treating and preventing malaria infections involving administering the pharmaceutical compositions to the subject.

RELATED APPLICATIONS

U.S. Provisional Patent Application 62/194,636, filed on 20 Jul. 2015and U.S. Provisional Patent Application 62/343,319, filed on 31 May 2016are related to this application and are hereby incorporated by referencein their entireties.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

Work resulting in this invention was funded by the United Statesgovernment under the terms of a VA Merit Review Grant awarded to Dr.Michael Riscoe by the United States Veterans Administration and GrantNumbers R56A1100569, R01A1100569, and PR130649 awarded by the NationalInstitutes of Health. The United States government has certain rights inthis invention.

FIELD

Generally, the field is small molecule therapeutics for use in treatinginfectious disease. More specifically, the field is anti-parasiticcompositions derived from quinolone-3-diarylethers.

BACKGROUND

Malaria remains an enormous global health problem Malaria remains one ofthe deadliest diseases in the world today, as it has been for thousandsof years. For each of the 1 million people killed each year, hundreds ofmillions more suffer from severe illness (1). Spread by mosquitoes fromperson to person malaria remains one of the most widespread infectiousdiseases of our time. There are five identified species of the parasiteresponsible for human malaria all belonging to genus Plasmodium. P.falciparum is the dominant species in sub-Saharan Africa, and isresponsible for the majority of the malaria-related deaths. P. vivax,known to be responsible for relapsing malaria, causes as much as 25-40%of the global malaria burden, whereas P. ovale, and P. malariaerepresent a small percentage of infections. A fifth species P. knowlesi,a species that infects subhuman primates, has Jed to human malaria, butthe exact mode of transmission remains unclear.

The impact of malaria is particularly devastating in sub-Saharan Africawhere its victims are primarily young children and pregnant women. Thissituation is worsened by the growing emergence of Plasmodium parasitesthat are resistant to multiple drugs (2). The list of drugs that arelosing potency against malaria includes the quinolines—chloroquine,quinine, and mefloquine; the antifolates—pyrimethamine and sulfadoxine;and the anti-respiratory combination of atovaquone (ATV) and proguanil.In SE Asia, treatment of multidrug resistant malaria relies solely onthe endoperoxide artesunate, leaving a razor thin wall of opposition tothe total collapse of malaria chemotherapy. One of the greatestchallenges in global health today is the development of a safe andaffordable drug for treatment and prevention of malaria (3).

SUMMARY

The antimalarial drug ELQ-300 is a selective sub-nanomolar inhibitor ofPlasmodium falciparum cytochrome bc1 complex. The effects of the drugare parasiticidal due to the requirement of cytochrome bc1 and thecoenzyme Q cycle for production of pyrimidines needed for DNA and RNAsynthesis. As a result, ELQ-300 exhibits an excellent parasitologicalprofile with potent activity against all life cycle stages of P.falciparum including liver, bloodstream, and vector stages.Unfortunately, the challenging physical-chemical characteristics ofELQ-300 limit its potential for clinical development, i.e., a highdegree of crystallinity (e.g., melting point>300° C.) and poor aqueoussolubility limit oral absorption to such a degree that it has beenimpossible to establish a therapeutic safety window. To address theissues of high crystallinity and poor water solubility we initiated aprodrug effort focusing primarily on carbonate ester prodrugs such asthe ethylcarbonate ester ELQ-337. The degree of crystallinity of thedrug was significantly reduced relative to ELQ-300 (i.e., melting pointfor ELQ-337=150° C.) and the oral bioavailability in mice and rats wasalso enhanced over ELQ-300. We now wish to disclose novelalkoxycarbonyloxyalkyl ester prodrugs of ELQ-300 (and similar4(1H)Quinolone-3-diarylether substituted derivatives such as ELQ-271,ELQ-316, and ELQ-400) with greatly reduced crystallinity as well asother features that suggest that they may be readily formulated forclinical use for treatment and prophylaxis against malaria as well asfor disease eradication.

Disclosed herein are compounds of the formula:

wherein X is halo and wherein R is an ester. In some examples, X isfluoro or chloro. In other examples, R is a carbonate ester. In stillmore examples, X is chloro and R is a carbonate ester selected frommethyl carbonate; ethyl carbonate; 2-methoxyethylcarbonate;2-(2-methoxyethoxy)ethyl carbonate; 2-(2-(2-methoxyethoxy)ethoxy)ethyl)carbonate; allyl carbonate; tert-butyl carbonate;((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) carbonate;((2-oxo-1,3-dioxolan-4-yl)methyl carbonate; 2,3-dihydroxypropylcarbonate; or 1,1-dioxidotetrahydrothiophen-3-yl carbonate. In stillfurther examples, X is fluoro and R is ethyl carbonate or pivalate. Inother examples, X is chloro and R is selected from isobutyrate,pivalate, or benzoate.

Disclosed herein are pharmaceutical compositions comprising atherapeutically effective amount of the compounds described herein. Thecomposition can further comprise polyethylene glycol or any otheracceptable additive.

Disclosed herein are uses of the pharmaceutical compositions describedherein for the treatment of malaria, toxoplasmosis, babesiosis,coccidiosis, cryptosporidiosis, cyclosporiasis, or isosporiasis in asubject. The pharmaceutical compositions can be administeredprophylactically or therapeutically. The pharmaceutical compositions canbe administered to a subject with a latent infection of any of theabove.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a plot of the disappearance of ELQ-331 over time in thepresence of pooled human microsomes (1 mg/ml of reaction.)

FIG. 2 is a plot of the conversion of ELQ-331 to ELQ-300 in the presenceof pooled human microsomes. The presence or absence of NADPH did notaffect the conversion rate.

DETAILED DESCRIPTION

Disclosed herein are ELQ-300 prodrugs comprising ester derivativesreplacing the ketone group at position 4 of the ELQ-300 quinoline.

Definitions

Unless specifically defined otherwise, the technical terms, as usedherein, have their normal meaning as understood in the art. Thefollowing explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.

“Administration of” and “administering” a compound refers to providing acompound, (such as a prodrug of a compound), or a pharmaceuticalcomposition comprising a compound or prodrug thereof to a subject. Thecompound or composition can be administered by another person to thesubject or it can be self-administered by the subject.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl,hexadecyl, eicosyl, tetracosyl and the like. A “lower alkyl” group is asaturated branched or unbranched hydrocarbon having from 1 to 6 carbonatoms (C₁₋₆alkyl). The term “alkyl” also includes cycloalkyl. The alkylgroup may be a “substituted alkyl” wherein one or more hydrogen atomsare substituted with a substituent such as halogen, cycloalkyl, alkoxy,amino, hydroxyl, aryl, or carboxyl.

Alkenyl refers to an unsaturated hydrocarbon group comprising at leastone carbon-carbon double bond.

The term “alkoxy” refers to an alkyl group attached to an oxygen atom toform an ether. The alkoxy group may be a “substituted alkoxy” whereinone or more hydrogen atoms are substituted with a substituent such ashalogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, or carboxyl.

The term “aryl” refers to any carbon-based aromatic group including, butnot limited to, benzene, naphthalene, phenyl, and oxazole. The term“aryl” also includes heteroaryl, which is defined as an aromatic groupthat has at least one heteroatom incorporated within the ring of thearomatic group. Examples of heteroatoms include, but are not limited to:nitrogen, oxygen, sulfur, and phosphorus. The aryl group can besubstituted with one or more groups including, but not limited to,alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ether,ketone, aldehyde, hydroxy, carboxylic acid, cyano, amido, haloalkyl,haloalkoxy, or alkoxy, or the aryl group can be unsubstituted.

The term “cycloalkyl” refers to a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,and cyclohexyl. The term “heterocycloalkyl group” is a cycloalkyl groupas defined above where at least one of the carbon atoms of the ring issubstituted with a heteroatom such as nitrogen, oxygen, sulfur, orphosphorus. “Derivative” refers to a compound or portion of a compoundthat is derived from or is theoretically derivable from a parentcompound.

The terms “halogenated alkyl” or “haloalkyl group” refer to an alkylgroup as defined above with one or more hydrogen atoms present on thesegroups substituted with a halogen (F, Cl, Br, I). For example, ahalomethyl group is a methyl group (—CH₃) with one or more halogenssubstituted for the hydrogens. A halomethyl group may include di- andtri-substituted halogens such as a trifluoromethyl group. A halogenatedether refers to a group with one or more hydrogen atoms present on anether, such as a methyl ether (—OCH₃), substituted with one or morehalogens. A halogenated ether may also be termed “halomethoxy” and thisgeneral term includes mono, di- and tri-substituted halogens on theether. For example, a trifluoromethyl ether has a formula of —OCF₃ andcan interchangeably be referred to as “trifluoromethoxy”. Similarly, adifluoromethoxy ether has the formula of —OCHF₂.

“Heterocycle” is a term that encompasses both heteroaryls andheterocycloalkyls. Heterocycles may be monocyclic or polycyclic rings.Exemplary heterocycles include, but are not limited to, azepinyl,aziridinyl, azetyl, azetidinyl, diazepinyl, dithiadiazinyl,dioxazepinyl, dioxolanyl, dithiazolyl, furanyl, isooxazolyl,isothiazolyl, imidazolyl, morpholinyl, oxetanyl, oxadiazolyl, oxiranyl,oxazinyl, oxazolyl, piperazinyl, pyrazinyl, pyridazinyl, pyrimidinyl,piperidyl, piperidino, pyridyl, pyranyl, pyrazolyl, pyrrolyl,pyrrolidinyl, thiatriazolyl, tetrazolyl, thiadiazolyl, triazolyl,thiazolyl, thienyl, tetrazinyl, thiadiazinyl, triazinyl, thiazinyl,thiopyranyl, furoisoxazolyl, imidazothiazolyl, thienoisothiazolyl,thienothiazolyl, imidazopyrazolyl, cyclopentapyrazolyl, pyrrolopyrrolyl,thienothienyl, thiadiazolopyrimidinyl, thiazolothiazinyl,thiazolopyrimidinyl, thiazolopyridinyl, oxazolopyrimidinyl,oxazolopyridyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl,imidazopyrazinyl, purinyl, pyrazolopyrimidinyl, imidazopyridinyl,benzimidazolyl, indazolyl, benzoxathiolyl, benzodioxolyl,benzodithiolyl, indolizinyl, indolinyl, isoindolinyl, furopyrimidinyl,furopyridyl, benzofuranyl, isobenzofuranyl, thienopyrimidinyl,thienopyridyl, benzothienyl, cyclopentaoxazinyl, cyclopentafuranyl,benzoxazinyl, benzothiazinyl, quinazolinyl, naphthyridinyl, quinolinyl,isoquinolinyl, benzopyranyl, pyridopyridazinyl and pyridopyrimidinylgroups.

The terms “treatment”, “treat” and “treating” refer to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition. As used herein, the terms “treatment”, “treat”and “treating,” with reference to a disease, pathological condition orsymptom, also refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, a reduction in the number of relapsesof the disease, an improvement in the overall health or well-being ofthe subject, or by other parameters well known in the art that arespecific to the particular disease.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs, forthe purpose of decreasing the risk of developing pathology.

A “therapeutic” treatment is a treatment administered to a subject whohas already begun to exhibit signs of a disease for the purpose ofslowing or reversing the pathology.

“Coadminister” means that each of at least two compounds areadministered during a time frame wherein the respective periods ofbiological activity overlap. Thus, the term includes sequential as wellas coextensive administration of two or more drug compounds.

The terms “pharmaceutically acceptable salt” or “pharmacologicallyacceptable salt” refers to salts prepared by conventional methods, andinclude basic salts of inorganic and organic acids, such as hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, andmandelic acid.

Pharmaceutically acceptable salts of the presently disclosed compoundsalso include those formed from cations such as sodium, potassium,aluminum, calcium, lithium, magnesium, zinc, and from bases such asammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine, diethylamine,piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammoniumhydroxide. These salts may be prepared by standard procedures, forexample by reaction of the free acid with a suitable organic orinorganic base. Any chemical compound recited in this specification mayalternatively be administered as a pharmaceutically acceptable saltthereof.

Pharmaceutically acceptable salts are also inclusive of the free acid,base, and zwitterionic forms. Descriptions of exemplary pharmaceuticallyacceptable salts can be found in Stahl and Wermuth, Eds., Handbook ofPharmaceutical Salts; Properties, Selection and Use, Wiley VCH (2008).When compounds disclosed herein include an acidic function such as acarboxy group, then suitable pharmaceutically acceptable cation pairsfor the carboxy group are well known to those skilled in the art andinclude alkaline, alkaline earth, ammonium, and quaternary ammoniumcations. Such salts are known to those of skill in the art. Foradditional examples of “pharmacologically acceptable salts,” see Bergeet al., J. Pharm. Sci. 66:1 (1977).

The term “subject” includes both human and veterinary subjects.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. For example, this may be the amount of a compounddisclosed herein useful in treating malaria in a subject. Ideally, atherapeutically effective amount of an agent is an amount sufficient toinhibit or treat the disease without causing substantial toxicity in thesubject. The therapeutically effective amount of an agent will bedependent on the subject being treated, the severity of the affliction,and the manner of administration of the therapeutic composition. Methodsof determining a therapeutically effective amount of the disclosedcompound sufficient to achieve a desired effect in a subject infectedwith a malaria parasite will be understood by those of skill in the artin light of this disclosure.

Synthesis of ELQ-300 Prodrugs

The term “prodrug” refers to any active or inactive compound that ismodified chemically through an in vivo physiological action, such ashydrolysis or metabolism, into an active compound followingadministration of the prodrug to a subject. The suitability andtechniques involved in making and using prodrugs are well known by thoseskilled in the art. For a general discussion of prodrugs involvingesters see Svensson and Tunek, Drug Metabolism Reviews 165 (1988), andBundgaard, Design of Prodrugs, Elsevier (1985).

The synthesis processes described herein can be monitored according toany suitable method known in the art. For example, product formation canbe monitored by spectroscopy, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C NMR), infrared spectroscopy,spectrophotometry (e.g., UV-visible), mass spectrometry, or bychromatography such as high performance liquid chromatograpy (HPLC) orthin layer chromatography.

Earlier attempts to develop a prodrug of ELQ-300 were unsuccessful—theprodrugs proved to be too unstable in physiological media to result insufficient bioavailability or too stable to be metabolized into activeELQ-300. For example, the 4-position acetyl ester (ELQ-370) ischemically unstable to mildly acidic conditions and even decomposesrapidly in methanol while the corresponding 4-oxo linkeddimethylcarbamate analog (ELQ-301) is stable to metabolism and displaysinferior in vivo efficacy compared to the parent compound ELQ-300.

Synthesis of ELQ-300 Carbonate Esters

Disclosed herein are O-linked esters and carbonates that are effectiveprodrugs of ELQ-300. ELQ-337 is the O-linked ethyl-carbonate of ELQ-300.O-linked carbonate esters of ELQs enhance oral delivery and efficacyagainst murine malaria. Placement of the promoiety at the 4-oxo-positionremoves the H-atom from the ring nitrogen thereby upsetting crystallattice formation. This is evidenced by a reduction of the melting pointfrom 314° C. for ELQ-300 to 160° C. for ELQ-337.

ELQ-337 is produced from ELQ-300 in one step, using sodium hydride intetrahydrofuran. Ethyl chloroformate is then added dropwise. Thereaction goes to completion in minutes upon the addition of thechloroformate forming one regioisomer in very high yield. ELQ-337 ischemically stable in 50/50 mixtures of methanol and water at pH 3 and 8overnight. Results are summarized in Scheme 1

Ester derivatives of ELQ-300, including carbonate esters are disclosed.

Ester Formation:

The R₁ group selected can result in the formation of any ester. Estersgenerally have the structure:

Carbonate esters generally have the structure:

wherein R₂ or R₃ can be any alkyl, substituted alkyl, alkenyl,substituted alkenyl, ether, substituted ether, aryl, substituted aryl,heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, orsubstituted heteroaryl.

The compounds disclosed herein have the general structure of Formula I:

wherein X is halo and R is ester. In particular examples X is chloro orfluoro. In still other examples, R is a carbonate ester. The carbonatecan be any carbonate ester including ethyl carbonate;2-methoxyethylcarbonate; 2-(2-methoxyethoxy)ethyl carbonate;2-(2-(2-methoxyethoxy)ethoxy)ethyl) carbonate; allyl carbonate;tert-butyl carbonate; ((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)carbonate; ((2-oxo-1,3-dioxolan-4-yl)methyl carbonate; and2,3-dihydroxypropyl carbonate. In still further examples, R is anon-carbonate ester. The non-carbonate ester can be any non-carbonateester including isobutyrate, pivalate, and benzoate groups.

As described herein, the definition of ester, particularly with regardto the R group of Formula I above does not encompass carbamates.Carbamates have the general structure:

Pharmaceutical Compositions

The compounds disclosed herein may be included in pharmaceuticalcompositions (including therapeutic and prophylactic formulations).Pharmaceutical compositions as described herein include one or morecompounds according to the present description. In addition to one ormore compounds as described herein, pharmaceutical compositionsaccording to the present disclosure may include one or more additionaltherapeutic agents, including, for example, one or more additionalantimalarial or anti-infective agents, antibiotics, anti-inflammatoryagents, or drugs that are used to reduce pruritus, such as anantihistamine. In preparing the pharmaceutical compositions, the one ormore compounds as described herein and, optionally, the one or moreadditional active agents, may be combined together with one or morepharmaceutically acceptable vehicles, salts, solubilizing agents (e.g.,co-crystals, lipids, or hydrophilic polymers) or carriers. Thepharmaceutical compositions described herein may be combined with orused simultaneously with one or more other therapeutic regimens orcompositions. Where one or more additional antimalarial oranti-infective agent is included in a pharmaceutical compositionaccording to the present invention, such agent(s) may be selected from,for example, quinolines, such as chloroquine, quinine, and mefloquine;the antifolates, such as pyrimethamine and sulfadoxine; theanti-respiratory agents atovaquone and/or proguanil, as well asinhibitors of parasite dihydro-orotate dehydrogenase (DHOD) such asDSM265.

Pharmaceutical compositions according to the present invention can beadministered to subjects by a variety of mucosal administration modes,including by oral, rectal, intranasal, intrapulmonary, or transdermaldelivery, or by topical delivery to other surfaces. Optionally, thecompositions can be administered by non-mucosal routes, including byintramuscular, subcutaneous, intravenous, intra-arterial,intra-articular, intraperitoneal, intrathecal, intracerebroventricular,or parenteral routes. In an embodiment, the compound can be administeredex vivo by direct exposure to cells, tissues or organs originating froma subject. To formulate the pharmaceutical compositions, the one or morecompounds can be combined with various pharmaceutically acceptableadditives, as well as a base or vehicle for dispersion of the compound.Such additives include, but are not limited to, pH control agents, suchas arginine, sodium hydroxide, glycine, hydrochloric acid, and citricacid. In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80 or medium chaintriacylglycerols such as myglyol 812), solubility enhancing agents (forexample, cyclodextrins and derivatives thereof), stabilizers (forexample, serum albumin), and reducing agents (for example, glutathione)can be included.

In preparing a pharmaceutical composition according to the presentdescription, the one or more compounds can be dispersed in a base orvehicle which can include a hydrophilic compound having a capacity todisperse the disclosed compound and any additives. The base can beselected from a wide range of suitable compounds, including but notlimited to, copolymers of polycarboxylic acids or salts thereof;carboxylic anhydrides (for example, maleic anhydride); with othermonomers (for example, methyl(meth)acrylate and acrylic acid);hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinylalcohol, polyvinylpyrrolidone, cellulose derivatives such ashydroxymethylcellulose and hydroxypropylcellulose; natural polymers,such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid;and nontoxic metal salts thereof.

A biodegradable polymer may be selected as a base or vehicle, such as,for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters and sucrosefatty acid esters may be employed as vehicles. Hydrophilic polymers andother vehicles can be used alone or in combination, and enhancedstructural integrity can be imparted to the vehicle by, for example,partial crystallization, ionic bonding, or cross-linking. The vehiclemay be provided in a variety of forms, including fluid or viscoussolutions, gels, pastes, powders, microspheres, and films for directapplication to a mucosal surface.

The one or more compounds may be combined with the base or vehicleaccording to a variety of methods, and release of the compound can bevia diffusion, disintegration of the vehicle, or associated formation ofwater channels. In some embodiments, the compound can be dispersed inmicrocapsules (microspheres) or nanoparticles prepared from a suitablepolymer, for example, 5-isobutyl-2-cyanoacrylate (see, for example,Michael et al, J. Pharmacy Pharmacol 43, 1-5, 1991), and dispersed in abiocompatible dispersing medium, which may provide sustained deliveryand biological activity over a protracted time.

In certain embodiments, the pharmaceutical compositions of thedisclosure can contain as pharmaceutically acceptable vehicles,substances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, andwetting agents, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,and triethanolamine oleate.

For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles may be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate.

Pharmaceutical compositions for administering the one or more compoundscan also be formulated as a solution, microemulsion, or other orderedstructure suitable for a high concentration of active ingredients. Thevehicle may be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol), and suitable mixtures thereof. Properfluidity for solutions may be maintained, for example, by the use of acoating such as lecithin, by the maintenance of a desired particle sizein the case of dispersible formulations, and by the use of surfactants.

In an embodiment, it may be desirable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol and sorbitol, or sodiumchloride in the composition. Prolonged absorption of the one or morecompounds may be obtained by including in the composition an agent whichdelays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the one or more compounds can be administered ina time release formulation, for example in a composition which includesa slow release polymer. These compositions may be prepared with vehiclesthat will protect against rapid release, for example, a controlledrelease vehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Controlled release binders suitable for use inaccordance with the disclosure include any biocompatible controlledrelease material which is inert to the active agent and which is capableof incorporating the compound and/or other biologically active agent.Numerous such materials are known in the art. Controlled-release bindersmay be materials that are metabolized slowly under physiologicalconditions following their delivery (for example, at a mucosal surface,or in the presence of bodily fluids).

Exemplary binders include, but are not limited to, biocompatiblepolymers and copolymers well known in the art for use in sustainedrelease formulations. Such biocompatible compounds are non-toxic andinert to surrounding tissues, and do not trigger significant adverseside effects, such as nasal irritation, immune response, orinflammation. They are metabolized into metabolic products that are alsobiocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity.

Exemplary polymers include polyglycolic acids and polylactic acids,poly(DL-lactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolicacid), and poly(L-lactic acid-coglycolic acid). Other usefulbiodegradable or bioerodable polymers include, but are not limited to,poly(epsilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid),poly(epsilon-caprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrylate), hydrogels such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) such as L-leucine, glutamicacid, L-aspartic acid, poly(ester urea), poly(2-hydroxyethylDL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate,polymaleamides, polysaccharides, and copolymers thereof.

Methods for preparing such formulations are well known to those skilledin the art (see, for example, Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,1978). Other useful formulations include controlled-releasemicrocapsules (U.S. Pat. Nos. 4,652,441 and 4,917,893), lacticacid-glycolic acid copolymers useful in making microcapsules and otherformulations (U.S. Pat. Nos. 4,677,191 and 4,728,721) and sustainedrelease compositions for water-soluble peptides (U.S. Pat. No.4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Dispersions may be prepared by incorporating the compoundand/or other biologically active agent into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated herein. In the case of sterile powders, methods ofpreparation include vacuum drying and freeze-drying which yields apowder of the compound plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The prevention of theaction of microorganisms can be accomplished by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, and thimerosal.

Methods of Treatment

The compounds and pharmaceutical compositions disclosed herein can beused for treating, inhibiting or preventing parasitic diseases, such asmalaria, caused by organisms such as Plasmodium sp., includingPlasmodium falciparum. Other examples of human or animal parasiticdiseases that may be treated using the compounds and pharmaceuticalcompositions disclosed herein include toxoplasmosis, amebiasis,giardiasis, leishmaniasis, trypanosomiasis, coccidiosis, andschistosomiasis, caused by organisms such as Toxoplasma sp., Eimeriasp., Babesia sp, or Theileria sp. Additional parasites that causemalaria include Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi,Plasmodium malariae, Plasmodium yoelii, and Plasmodium berghei.

In particular embodiments, the compounds and compositions disclosedherein can be administered to a subject to prevent or inhibitdrug-resistant malaria such as chloroquine-resistant malaria ormultidrug-resistant malaria that is caused by organisms harboringresistance to chloroquine, quinine, mefloquine, pyrimethamine, dapsone,atovaquone, P. falciparum DHOD inhibitors such as DSM265 (Coteron J M etal, J Med Chem 54, 5540-5561 (2011); incorporated by reference herein)or any other available anti-malarial drug.

In further embodiments, the compounds and pharmaceutical compositionsdisclosed herein can be coadministered with another pharmaceuticallyactive compound. For example, the compounds may be coadministered withquinine, chloroquine, atovaquone, proguanil, primaquine, amodiaquine,mefloquine, piperaquine, artemisinin, artesunate, endoperoxidases,methylene blue, pyrimethamine, sulfadoxine, artemether-lumefantrine(Coartem®), dapsone-chlorproguanil (LAPDAP®), artesunate, quinidine,clopidol, pyridine/pyridinol analogs, 4(1H)-quinolone analogs,dihydroartemisinin, a mixture of atovaquone, proguanil, an endoperoxide,an acridone as disclosed in WO 2008/064011, another 3-aryl quinoline asdisclosed in WO 2010/059633, or any combination or mixtures of these,whether administered separately or in a single pharmaceuticalcomposition.

In accordance with the various treatment methods of the disclosure, thecompound can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the compound and/or other biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

Typical subjects intended for treatment with the compounds, compositionsand methods of the present disclosure include humans, as well asnon-human primates and other animals such as companion animals,livestock animals, animals used in models of parasitic infection, oranimals used in pharmaceutical testing, such as pharmacokinetics andtoxicological testing, including mice, rats, rabbits, and guinea pigs.

To identify subjects for prophylaxis or treatment according to themethods of the disclosure, accepted screening methods are employed todetermine risk factors associated with a parasitic infection todetermine the status of an existing disease or condition in a subject.These screening methods include, for example, preparation of a bloodsmear from an individual suspected of having malaria. The blood smear isthen fixed in methanol and stained with Giemsa and examinedmicroscopically for the presence of Plasmodium infected red blood cells.These and other routine methods allow a clinician to select patients inneed of therapy using the methods and pharmaceutical compositions of thedisclosure.

The administration of the disclosed compounds and pharmaceuticalcompositions can be for prophylactic or therapeutic purposes or to blocktransmission of disease. When provided prophylactically, the compound isadministered to a subject in advance of a symptom. The prophylacticadministration of the compound serves to prevent or amelioratesubsequent disease process or to achieve disease eradication. Whenprovided therapeutically, the compound is administered to a subject ator after the onset of a symptom of disease or infection.

For prophylactic and therapeutic purposes, the compound orpharmaceutical composition may be administered to the subject orally orin a single bolus delivery, via continuous delivery (for example,continuous transdermal, mucosal or intravenous delivery) over anextended time period, or in a repeated administration protocol (forexample, by an hourly, daily or weekly, repeated administrationprotocol). The therapeutically effective dosage of the compound may beprovided as repeated doses within a prolonged prophylaxis or treatmentregimen to yield clinically significant results to alleviate one or moresymptoms or detectable conditions associated with a targeted disease orcondition as set forth herein.

Determination of effective dosages in this context may be based onanimal model studies followed up by human clinical trials and may beguided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, avian, porcine, feline, non-human primate, and other acceptedanimal model subjects known in the art. Alternatively, effective dosagesmay be determined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, calculations and adjustmentscan be required to determine an appropriate concentration and dose toadminister a therapeutically effective amount of the compound (forexample, amounts that are effective to elicit a desired immune responseor alleviate one or more symptoms of a targeted disease). In certainembodiments, an effective amount or effective dose of the compound maysimply inhibit or enhance one or more selected biological activitiescorrelated with a disease or condition, as set forth herein, for eithertherapeutic or diagnostic purposes.

The actual dosage of the compound may vary according to factors such asthe disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms, andsusceptibility factors), time and route of administration, other drugsor treatments being administered concurrently, as well as the specificpharmacology of the compound for eliciting the desired activity orbiological response in the subject. Dosage regimens can be adjusted toprovide an optimum prophylactic or therapeutic response.

A therapeutically effective amount may be one in which any toxic ordetrimental side effects of the compound and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A non-limiting range for a therapeutically effectiveamount of a compound and/or other biologically active agent within themethods and compositions of the disclosure is about 0.01 mg/kg bodyweight to about 100 mg/kg body weight, such as about 0.05 mg/kg to about50 mg/kg body weight, or about 0.5 mg/kg to about 5 mg/kg body weight.

The dosage may be varied to maintain a desired concentration at a targetsite (for example, the lungs or systemic circulation). Higher or lowerconcentrations can be selected based on the mode of delivery, forexample, trans-epidermal, rectal, oral, pulmonary, or intranasaldelivery versus intravenous or subcutaneous delivery. Dosage can also beadjusted based on the release rate of the administered formulation, forexample, of an intrapulmonary spray versus powder or sustained releaseoral versus injected particulate or transdermal delivery formulations.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or devices and consumables that facilitate theadministration the same for use in the prevention and treatment ofdiseases and other conditions in mammalian subjects.

In an embodiment, the compound may be formulated in a pharmaceuticalcomposition for delivery to a subject. In such embodiments,pharmaceutical compositions according to the present description may beused. The compound or composition within which it is formulated may becontained in a bulk dispensing container or unit or multiunit dosageform. Optional dispensers can be provided, for example, a pulmonary orintranasal spray applicator. Packaging materials optionally include alabel or instruction indicating for what treatment purposes and/or inwhat manner the pharmaceutical agent packaged therewith can be used.

In an embodiment, the method of treating a Plasmodium infectioncomprises administering a therapeutically effective amount of acompound. The compound may be administered orally, subcutaneously,intravenously, or intramuscularly to a subject suffering from or at riskof suffering from a Plasmodium infection.

Mechanism of Action of ELQ-300 Prodrugs

The chemical structures of the prodrugs ELQ-330 and ELQ-331 are shown inScheme 1 below along with the structure of the parent drug ELQ-300. Itis well established that alkoxycarbonyloxyalkyl ester prodrugs serve asneutral lipophilic prodrugs of pharmaceutical agents. Consider forexample the clinical drug Tenofovir Disoproxil (FIG. 2) which containstwo alkoxycarbonyloxyalkyl ester promoieties attached to a centralphosphonate residue. ELQ-330 and ELQ-331 were formed by reaction ofELQ-300 with either chloro-methyl-isopropylcarbonate (ELQ-330) orchloro-methyl-ethylcarbonate (ELQ-331).

The development of ELQ-300 for clinical use is hindered by relativelypoor water solubility that is linked to a high degree of crystallinity.One simple measure that can be used to compare crystal lattice strengthis the melting point. Pure ELQ-300 has a melting point of >300° C.Disclosed herein are compounds with an alkoxycarbonate ester at position4 of the quinoline ring system have significantly reduced crystallattice energy compared to ELQ-300 as evidenced by an impressive drop inthe melting point: 99.7-99.9/ELQ-330; 103.5-103.7° C./ELQ-331; and135.0-135.7° C./ELQ-387. Without being bound by theory, reducingcrystallinity means that the alkoxycarbonate ester prodrugs can have areduced tendency for re-crystallization in the intestines prior toabsorption. As a consequence, this subclass of ELQ prodrugs, i.e., thealkoxycarbonate esters, can be used to improve oral bioavailability andbloodstream exposures to the active parent ELQs (e.g., ELQ-271, ELQ-300,ELQ-316, and ELQ-400).

The potential for hepatic P450-dependent metabolism of ELQ-331 at 11 μMwas assessed using pooled human liver microsomes in the presence of anNADPH regenerating system. The loss of substrate was monitored byLC/MS/MS. Midazolam was monitored as a well characterized positivecontrol. Midazolam represents a drug with comparatively low metabolicstability. Incubations were conducted in the absence of the NADPHregenerating system to monitor for potential P450-independentmetabolism. In the absence of NADPH we did not observe significantmetabolism of the midazolam control however it was rapidly metabolizedin the presence of NADPH with a t112 value of 2.6 minutes. In thepresence of 1 mg/ml of pooled human microsomes and NADPH regeneratingsystem we observed a linear conversion of ELQ-331 to ELQ-300 throughoutthe first 20 minutes of reaction time. The t_(1/2) value recorded forELQ-331 was 37.7 minutes in the presence or absence of NADPH. That theconversion rate of the prodrug ELQ-331=>ELQ-300 did not vary between thesamples with and without NADPH indicates that host esterases areprimarily responsible for enzymatic production of ELQ-300 from ELQ-331(FIGS. 1 and 2)

EXAMPLES

The following examples are for illustration only. In light of thisdisclosure, those of skill in the art will recognize that variations ofthese examples and other examples of the disclosed invention be possiblewithout undue experimentation.

Example 1—ELQ-331 Synthesis

ELQ-300 (0.85 g, 1.8 mmol), tetrabutylammonium iodide (1.33 g, 3.6 mmol)and potassium carbonate (0.50 g, 3.6 mmol) were dissolved anhydrousdimethylformamide (8 ml) in a flame-dried round bottom flask at 60° C.under inert atmosphere. Chloromethyl ethyl carbonate (0.5 g, 3.6 mmol)was added dropwise and the reaction stirred under inert atmosphere at60° C. for two hours, at which point reaction completion was confirmedby thin layer chromatography. After cooling to room temperature, thereaction solvent was removed under reduced pressure and the mixturetaken up in water (10 ml) and extracted with dichloromethane (3×20 ml).Combined organic layers were washed with brine (10 ml), dried overMgSO₄, and the dichloromethane evaporated under reduced pressure. Theresulting crude product was purified by flash chromatography (EtOAc/DCM)to yield the title compound, ELQ-331, as a white crystalline solid (560mg, 54%). ¹H NMR (400 MHz, DMSO-d₆): δ=7.98 (s, 1H), 7.57 (s, 1H), 7.44(m, 4H), 7.21 (m, 4H), 5.76 (s, 2H), 5.35 (s, 2H), 4.03 (s, 3H), 2.44(s, 3H), 1.11 (t, 3H, J=7.1 Hz); M.P. (° C.): 103.5-103.7.

Example 2—Characterization of ELQ-331 by Gas Chromatography-MassSpectrometry (gc-ms)

ELQ-331 was characterized by gc-ms on an Agilent 5977A MSD/5890B gaschromatography system. The instrument was equipped with an Agilent J&WGC column with stationary phase HP-5MS with overall dimensions of 30m×0.250 mm×0.25 micrometers with helium as the inert carrier gas. Thetemperature gradient was 200-300° C. at 30° C./min.

Example 3—ELQ-387 Synthesis

Tetrabutyl-ammonium iodide (0.15 g, 0.42 mmol), potassium carbonate(0.06 g, 0.42 mmol), and 1-chloroethyl ethyl carbonate (0.06 mL, 0.42mmol) were dissolved in anhydrous dimethylformamide (5 mL) in aflame-dried round bottom flask at 70° C. under inert atmosphere. ELQ-300(0.10 g, 0.21 mmol) was added and the reaction stirred under inertatmosphere at 70° C. for four hours, until complete by thin layerchromatography. The reaction was cooled to room temperature and thereaction solvent evaporated under temperature and the reaction solventevaporated under reduced pressure. The mixture was taken up in water (10ml) and extracted with dichloromethane (3×15 mL). Combined organiclayers were washed with brine (15 mL) and concentrated. Purification bysilica column chromatography (EtOAc/DCM) yielded the title compound,ELQ-387, as a white crystalline solid (39 mg, 32%). ¹H NMR (400 MHz,DMSO-d₆): δ=8.03 (s, 1H), 7.54 (s, 1H), 7.44 (m, 3H), 7.25 (m, 2H), 7.20(m, 2H), 5.83 (q, 1H, J=5.4 Hz), 4.02 (s, 3H), 3.79 (m, 2H), 2.44 (s,3H), 1.19 (d, 3H, J=5.3 Hz), 0.88 (t, 3H, J=7.1 Hz); M.P. (° C.):135.0-135.7.

Example 4—In Vitro Antiplasmodial Activity of ELQ-330, ELQ-331, andELQ-387 vs. Chloroquine Sensitive (D6) and Resistant (Dd2, Tm90.C2B)Strains of Plasmodium falclparum

ELQ-330, ELQ-331 and ELQ-387 were evaluated for anti-plasmodial activityby the fluorescence based SYBR green assay developed in our lab andpublished in 2004. Briefly, experiments were set up in triplicate in96-well plates (Costar, Corning) with 2-fold dilutions of each drugacross the plate in a total volume of 100 μL and at a final red bloodcell concentration of 2% (v/v). The dilution series was initiated at aconcentration of 1 μM and the experiment was repeated beginning with alower initial concentration for those compounds in which the IC₅₀ valuewas below 10 nM. Automated pipetting and dilution was carried out withthe aid of a programmable Precision 2000 robotic station (BioTek,Winooski, Vt.). An initial parasitemia of 0.2% was obtained by additionof normal uninfected red cells to a stock culture of asynchronousparasite infected red cells (PRBC). The plates were incubated for 72 hat 37° C. in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂. After thisperiod, the SYBR Green I dye-detergent mixture (100 UL) was added andthe plates were incubated at room temperature for an hour in the darkand then placed in a 96-well fluorescence plate reader (SpectramaxGemini-EM, Molecular Diagnostics) for analysis, with excitation andemission wavelength bands centered at 497 and 520 nm, respectively. Thefluorescence readings were plotted against the logarithm of the drugconcentration, and curve fitting by nonlinear regression analysis(GraphPad Prism software) yielded the drug concentration that produced50% of the observed decline relative to the maximum readings indrug-free control wells (IC₅₀). Chloroquine was used as an internalcontrol to establish zero percent viability and cross-resistance.

IC₅₀ values are presented in Table 1. As shown the IC₅₀ values foralkoxycarbonyloxyalkyl ester prodrugs ELQ-330 and ELQ-387 aresignificantly higher than for the parent molecule ELQ-300 against allthree tested strains. These results indicate that while P. falciparuminfected red cells apparently have the enzymic capacity to break downthe prodrugs to release ELQ-300 it would appear that for thesealkoxycarbonyloxyalkyl ester are poorly processed by parasite encodedesterases. It is both interesting and significant that the IC₅₀ valuesfor ELQ-331 are quite similar to the ELQ-300 values, thereby indicatingthat this prodrug is more effectively converted to ELQ-300 by P.falciparum esterases. Taken together and because it appears that ELQ-331is more efficiently converted to ELQ-300 by both host as well asparasite esterases we hypothesized that this drug would have superiorefficacy in vivo.

TABLE 1 Antiplasmodial activities for standard antimalarials(Chloroquine and Atovaquone) and ELQ-300 and the esterase sensitiveprodrug ELQ-337 against drug sensitive (06) and multidrug resistant (Dd2and Tm90:C2B) strains of P. falciparum. IC₅₀, nM, IC₅₀, nM, IC₅₀, nM, P.falciparum P. falciparum P. falciparum strain Drug strain D6⁸ strainDd2⁸ Tm90-C2B⁸ Chloroquine 10 137 98 Atovaquone 0.2 0.2 >250 ELQ-300 6 62 ELQ-330 62 38 47 ELQ-331 6 8 4 ELQ-387 326 141 231 ⁸IC₅₀ = The drugconcentration that decreases parasite proliferation by 50% relative tocontrol (no-drug) values. D6 is sensitive to chloroquine while Dd2 andTm90-C2B are resistant to chloroquine. Tm90-C2B Is also resistant to theantirespiratory drug atovaquone.

Example 5—In Vivo Efficacy of Alkoxycarbonyloxyalkyl Ester ProdrugsELQ-330 and ELQ-331 Against the Blood Stage of Murine Malaria Infection

Typically, antimalarial drugs are provided over the course of a 3 to 4day regimen. Such multi-dose schedules are sub-optimal because it simplymay not be feasible in the field where resources are often limited anddosing schedules may vary. Ideally drugs could be delivered in a singledose regimen that can be directly monitored to ensure compliance.Currently there are no drugs in clinical use for treatment of malariawith sufficient potency and safety to deliver cures following a singleoral dose.

We evaluated ELQ-330 and ELQ-331 for their potential to cure mice of apatent malaria infection in a single dose, i.e., single dose cure (SDC),and compared our findings to the direct administration of the parentdrug ELQ-300. As described above, mice (female, CF1, Charles River Labs)were infected intravenously with 10⁵ P. yoelii (Kenya strain, MR4MRA-428/Murine LDH Elevating Virus-Free) parasitized erythrocytes from adonor animal. Drug administration commenced the day after the animalswere inoculated (Day 1). The test compounds were dissolved in PEG-400and administered by oral gavage once. On the 5th day blood films wereprepared and the extent of parasitemia was determined by microscopicexamination of Giemsa stained smears. Animals remaining parasite free 30days after the last drug dose were considered cured of their infection.

In vivo studies were carried out as described above with ELQ-300 as aninternal control. As previously published, while ELQ-300 is highlyeffective in low multi-dose regimens its poor aqueous solubility andhigh crystallinity prevent it from being useful as a single dosecurative agent. In this experiment carried out at oral doses in therange of 1 to 20 mg/kg, ELQ-300 suppressed parasitemia completelyhowever recrudescence occurred within two weeks of dosing. In comparisonboth ELQ-330 and ELQ-331 proved highly effective against murine malariaand superior to the parent drug ELQ-300. The lowest fully protectivesingle-dose cure for ELQ-330 was achieved with an oral dose of 5 mg/kgand for ELQ-331 the lowest observable SDC was 2.5 mg/kg. Evaluation ofELQ-387 is currently being evaluated in this model. Taken together It isclear that the 4-position alkoxycarbonate ester derivatives of ELQ-300are highly effective prodrugs that may be formulated for clinical use asantimalarial agents. Similar prodrug variants of other ELQs withclinical or veterinary potential should be more effective than thecorresponding parent molecule, e.g., ELQ-271, ELQ-300, ELQ-316, andELQ-400 etc., for treatment of malaria and other parasitic diseasesincluding malaria (falciparum, vivax, ovale, knowlesi, and malariae),toxoplasmosis, babesiosis, coccidiosis, theileria, and other diseasescaused by Apicomplexan parasites.

Example6—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylethyl carbonate (ELQ-337)

To a flame dried 50 mL round bottom flask was added 0.5 g ELQ-300 (1.05mmol, 1 eq), 84 mg sodium hydride (60% disp., 2.1 mmol, 2 eq) andanhydrous THF 5 mL. The resulting suspension was heated and stirred at60° C. under argon atmosphere for 30 mins or until a clear solution wasobtained. The reaction was removed from heat and 200 μL Ethylchloroformate (2.1 mmol, 2 eq) was added dropwise via a syringeresulting in an immediate precipitation of white solids. The suspensionwas stirred for 5 mins and then quenched by dropwise addition of water.The reaction mixture was diluted with water (5 mL) and extracted withethyl acetate (3×5 mL). The organic layer was washed with brine (5 mL)and dried over MgSO₄. The residue after evaporation was recrystallized(DCM, hexanes) to give 0.558 g ELQ-337(6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylethyl carbonate) (97%) as white microcrystals. ¹H NMR (400 MHz, CDCl₃) δ7.89 (s, 1H), 7.50 (s, 1H), 7.32-7.27 (m, 2H), 7.23 (d, J=8.4 Hz, 2H),7.15-7.01 (m, 4H), 4.16 (q, J=7.1 Hz, 2H), 4.06 (s, 3H), 2.54 (s, 3H),1.22 (t, J=7.1 Hz, 3H).

Example7—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl(2-methoxyethyl)carbonate (ELQ-354)

ELQ-354 was prepared according to the method of Example 6 except that 2eq. 2-methoxyethyl chloroformate was used in place of ethylchloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (bs, 1H), 7.52 (s, 1H),7.29 (d, J=8.7 Hz, 2H), 7.24 (d, J=9.0 Hz, 2H), 7.14-7.04 (m, 4H),4.31-4.20 (m, 2H), 4.07 (s, 4H), 3.57-3.49 (m, 2H), 3.37 (s, 3H), 1.25(s, 3H).

Example8—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl(2-(2-methoxyethoxy)ethyl) carbonate (ELQ-362)

ELQ-362 was prepared according to the method of Example 6 except that 2eq. 2-(2-methoxyethoxy)ethyl chloroformate was used in place of ethylchloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (s, 1H), 7.52 (s, 1H),7.33-7.27 (m, 2H), 7.23 (s, 2H), 7.15-7.05 (m, 4H), 4.30-4.19 (m, 2H),4.06 (d, J=9.9 Hz, 4H), 3.68-3.58 (m, 4H), 3.57-3.50 (m, J=6.0, 3.0 Hz,2H), 3.37 (s, 3H), 1.25 (s, 3H).

Example9—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl(2-(2-(2-methoxyethoxy)ethoxy)ethyl) carbonate (ELQ-363)

ELQ-363 Was prepared according to the method of Example 6 above exceptthat 2 eq. 2-(2-(2-methoxyethoxy)ethoxy)ethyl chloroformate was used inplace of ethyl chloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.89 (s, 1H),7.49 (bs, 1H), 7.28 (d, J=8.7 Hz, 2H), 7.24 (d, J=8.5 Hz, 2H), 7.13-7.06(m, 4H), 4.27-4.22 (m, 2H), 4.06 (s, 3H), 3.68-3.60 (m, 8H), 3.54 (dd,J=5.7, 3.6 Hz, 2H), 3.37 (s, 3H), 1.25 (s, 3H).

Example 10—allyl(6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl)carbonate (ELQ-359)

ELQ-359 was prepared according to the method of Example 6 above exceptthat 2 eq. allyl chloroformate in was used place of ethyl chloroformate.¹H NMR (400 MHz, CDCl₃) δ 7.89 (s, 1H), 7.52 (s, 1H), 7.28 (d, J=8.7 Hz,2H), 7.23 (d, J=8.4 Hz, 2H), 7.14-7.02 (m, 4H), 5.79 (ddd, J=22.7, 11.0,5.7 Hz, 1H), 5.31-5.26 (m, 1H), 5.23 (dd, J=10.6, 1.2 Hz, 1H), 4.58 (dt,J=5.7, 1.2 Hz, 2H), 4.06 (s, 3H), 2.54 (s, 3H).

Example11—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylisobutyrate (ELQ-375)

ELQ-375 was prepared according to the method of Example 6 above exceptthat 2 eq. isobutyryl chloride was used in place of ethyl chloroformate.¹H NMR (400 MHz, CDCl₃) δ 8.50 (s, 1H), 7.86 (s, 1H), 7.25-7.02 (m, 8H),4.19 (s, 3H), 2.88 (s, 3H), 2.71 (dq, J=13.8, 7.0 Hz, 1H), 1.08 (d,J=7.0 Hz, 6H).

Example12—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylpivalate (ELQ-357)

ELQ-357 was prepared according to the method of Example 6 above exceptthat 2 eq. trimethylacetyl chloride was used in place of ethylchloroformate. ¹H NMR (400 MHz, CDCl₃) δ 8.35 (s, 1H), 7.78 (s, 1H),7.25-7.01 (m, 8H), 4.17 (s, 3H), 2.80 (s, 3H), 1.15 (s, 9H).

Example13—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylbenzoate (ELQ-379)

ELQ-379 was prepared according to the method of Example 6 above except 2eq. Benzoyl chloride was used in place of ethyl chloroformate. ¹H NMR(400 MHz, CDCl₃) δ 8.00 (dd, J=8.3, 1.2 Hz, 2H), 7.81 (s, 1H), 7.66 (dd,J=10.6, 4.4 Hz, 1H), 7.53 (s, 1H), 7.49 (t, J=7.8 Hz, 2H), 7.30 (d,J=8.7 Hz, 2H), 7.03 (dd, J=31.9, 8.5 Hz, 4H), 6.83-6.70 (m, 2H), 4.07(s, 3H), 2.58 (s, 3H).

Example 14—tert-butyl(6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl)carbonate (ELQ-358)

ELQ-358 was prepared according to the method of Example 6 above exceptthat 2 eq. di-tert-butyl dicarbonate was used in place of ethylchloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (s, 1H), 7.49 (s, 1H),7.32-7.28 (m, 2H), 7.25-7.20 (m, 2H), 7.13-7.03 (m, 4H), 4.06 (s, 3H),2.54 (s, 3H), 1.37 (s, 9H)

Example15—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) carbonate (ELQ-374)

ELQ-374 was prepared according to the method of Example 6 above exceptthat 2 eq. (2,2-dimethyl-1,3-dioxolan-4-yl)methyl chloroformate was usedin place of ethyl chloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (s, 1H),7.52 (s, 1H), 7.28 (d, J=8.6 Hz, 2H), 7.24 (d, J=9.3 Hz, 2H), 7.10 (dd,J=8.8, 1.7 Hz, 4H), 4.23-4.08 (m, 3H), 4.06 (s, 3H), 4.01 (dd, J=8.6,6.3 Hz, 1H), 3.64 (dd, J=8.6, 5.3 Hz, 1H), 2.54 (s, 3H), 1.40 (s, 3H),1.37 (s, 3H).

Example16—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl(2,3-dihydroxypropyl) carbonate (ELQ-376)

ELQ-376 was prepared according to the method of Example 6 above, exceptthat 2 eq. (2-oxo-1,3-dioxolan-4-yl)methyl chloroformate was used inplace of ethyl chloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H),7.50 (s, 1H), 7.27 (t, J=8.0 Hz, 4H), 7.17-7.05 (m, 4H), 4.79 (ddt,J=8.4, 6.2, 4.1 Hz, 1H), 4.48 (t, J=8.7 Hz, 1H), 4.31 (qd, J=12.4, 4.0Hz, 2H), 4.07 (t, J=7.5 Hz, 1H), 4.06 (s, 3H), 2.54 (s, 3H).

Example 17—6-chloro-7-methoxy-2methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl((2-oxo-1,3-dioxolan-4-yl)methyl) carbonate (ELQ-373)

ELQ-373 was prepared from 6-chloro-7-methoxy-2-methyl-3-(4-(4(trifluoromethoxy)phenoxy)phenyl) quinolin-4-yl((2,2-dimethyl-1,3-dioxolan-4-yl)methyl) carbonate upon stirring in 2MHCl for 12 h. ¹H NMR (400 MHz, CDCl₃) δ 7.79 (s, 1H), 7.44 (s, 1H),7.27-7.11 (m, 4H), 7.11-7.00 (m, 4H), 4.72 (ddt, J=8.3, 6.2, 4.1 Hz,1H), 4.41 (t, J=8.7 Hz, 1H), 4.24 (qd, J=12.3, 3.9 Hz, 2H), 4.00 (s,3H), 4.04-3.93 (m, 1H), 2.48 (s, 3H).

Example18—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-yl(1,1-dioxidotetrahydrothiophen-3-yl) carbonate (ELQ-355)

ELQ-355 was prepared according to the method of Example 6 above, exceptthat 2 eq. 1,1-dioxidotetrahydrothiophen-3-yl chloroformate was used inplace of ethyl chloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.88 (s, 1H),7.52 (s, 1H), 7.39-7.27 (m, J=2.6 Hz, 4H), 7.12 (dd, J=13.0, 8.9 Hz,4H), 5.36-5.22 (m, 1H), 4.07 (s, 3H), 3.30 (dd, J=14.7, 6.5 Hz, 1H),3.12 (dd, J=9.6, 5.8 Hz, 2H), 2.91 (d, J=14.6 Hz, 1H), 2.56 (s, 3H),2.50-2.38 (m, 1H), 2.35-2.22 (m, 1H).

Example19—6-chloro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylmethyl carbonate (ELQ-336)

ELQ-336 was prepared according to the method of Example 6 above, exceptthat 2 eq. methyl chloroformate was used in place of ethylchloroformate. ¹H NMR (400 MHz, CDCl₃) δ 7.88 (s, 1H), 7.51 (s, 1H),7.30-7.27 (m, 2H), 7.24 (d, J=8.4 Hz, 2H), 7.13-7.05 (m, 4H), 4.06 (s,3H), 3.75 (s, 3H), 2.54 (s, 3H).

Example20—6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylethyl carbonate (ELQ-334)

ELQ-334 was prepared according to the method of Example 6 above from6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-oland ethyl chloroformate. ¹H-NMR (400 MHz; CDCl₃): δ 7.52 (d, J=8.0 Hz,1H), 7.48 (d, J=11.2 Hz, 1H), 7.30-7.27 (m, 2H), 7.24-7.22 (m, 2H),7.12-7.06 (m, 4H), 4.15 (q, J=7.1 Hz, 2H), 4.04 (s, 3H), 2.53 (s, 3H),1.22 (t, J=7.1 Hz, 3H). ¹⁹F-NMR (376 MHz; CDCl₃): δ −58.26 (s, 1F),−131.68 (t, J=9.9 Hz).

Example21—6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-ylpivalate (ELQ-377)

ELQ-377 was prepared according to the method of Example 6 above from6-fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4-oland trimethylacetyl chloride ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J=8.0Hz, 1H), 7.28 (s, 1H), 7.25-7.19 (m, 4H), 7.12-7.00 (m, 4H), 4.04 (s,3H), 2.49 (s, 3H), 1.14 (s, 9H).

Example 22—Efficacy of ELQ-337 In Vitro and In Vivo

In vitro experiments show that the intrinsic antiplasmodial activity ofethylcarbonate ester ELQ-337 is indistinguishable from ELQ-300, withIC₅₀ values against all test strains in the low to sub-nanomolar range.In vivo experiments using the 4-day suppression test protocol (dosing on4 sequential days with smears on Day 5) vs. P. yoelii infected mice showthat the action profile is unchanged at lower doses needed for ED₅₀(0.02 mg/kg/d), ED₉₀ (0.05 mg/kg/d), ED₉₉ (0.075 mg/kg/d), andnon-recrudescence dose remains impressive (0.3 to 1 mg/kg/d).Importantly and unlike ELQ-300 (at any dose), ELQ-337 provided 4/4single dose cures (SDC) at doses as low as 3 mg/kg (1 mg/kg failed in4/4 animals on Day 12).

TABLE 1 Efficacy of exemplary compounds. Lowest fully P. effectivefalciparum single dose strain D6, cure P. yoelii Code Chemical Structurec_(log)P IC₅₀, nM (mg/kg) ELQ-300

5.66 3.1 >20*  ELQ-336

8.16 2.5   4 ELQ-337

8.5  2.5   3 ELQ-354

7.2  3.0   3 ELQ-355

5.8  3.9   4 ELQ-357

8.5  3.5   4 ELQ-358

8.3  6.0 >4 ELQ-359

7.9  2.5   4 ELQ-373

6.2  3.1   4 ELQ-374

8.0  2.7   4 ELQ-375

8.1  8.2   4 ELQ-379

9.4  2.5 ND

1-12. (canceled)
 13. A method of treating or inhibiting malaria in ahuman, the method comprising administering to the human in need thereofa therapeutically effective amount of a compound selected from the groupof:

or a pharmaceutically acceptable salt thereof.
 14. The method of claim13, wherein the malaria is chloroquine-resistant malaria.
 15. The methodof claim 13, wherein the malaria is multidrug-resistant malaria.
 16. Themethod of claim 15, wherein the multidrug-resistant malaria is caused byresistance to two or more agents selected from the group of chloroquine,quinine, mefloquine, pyrimethamine, dapsone, atovaquone, or a P.falciparum DHOD inhibitor; or a pharmaceutically acceptable saltthereof.
 17. The method of claim 13, wherein the method furthercomprises co-administering with the compound of claim 1, or apharmaceutically acceptable salt thereof, to the human in need thereof atherapeutically effective amount of one or more compounds selected fromthe group of quinine, chloroquine, atovaquone, proguanil, primaquine,amodiaquine, mefloquine, piperaquine, artemisinin, artesunate, methyleneblue, pyrimethamine, sulfadoxine, artemether-lumefantrine,dapsone-chlorproguanil, artesunate, quinidine, clopidol, andihydroartemisinin, or a pharmaceutically acceptable salt thereof. 18.The method of claim 13 comprising administering the compound, or apharmaceutically acceptable salt thereof, to the human in need thereofprophylactically.
 19. The method of claim 13, wherein the human in needthereof has a latent malaria infection.
 20. A method of treating orinhibiting malaria in a human, the method comprising administering tothe human in need thereof a therapeutically effective amount of acompound of the formula:

or a pharmaceutically acceptable salt thereof.
 21. The method of claim20, wherein the malaria is chloroquine-resistant malaria.
 22. The methodof claim 20, wherein the malaria is multidrug-resistant malaria.
 23. Themethod of claim 22, wherein the multidrug-resistant malaria is caused byresistance to two or more agents selected from the group of chloroquine,quinine, mefloquine, pyrimethamine, dapsone, atovaquone, or a P.falciparum DHOD inhibitor; or a pharmaceutically acceptable saltthereof.
 24. The method of claim 20, wherein the method furthercomprises co-administering with the compound of claim 1, or apharmaceutically acceptable salt thereof, to the human in need thereof atherapeutically effective amount of one or more compounds selected fromthe group of quinine, chloroquine, atovaquone, proguanil, primaquine,amodiaquine, mefloquine, piperaquine, artemisinin, artesunate, methyleneblue, pyrimethamine, sulfadoxine, artemether-lumefantrine,dapsone-chlorproguanil, artesunate, quinidine, clopidol, andihydroartemisinin, or a pharmaceutically acceptable salt thereof. 25.The method of claim 20 comprising administering the compound, or apharmaceutically acceptable salt thereof, to the human in need thereofprophylactically.
 26. The method of claim 20, wherein the human in needthereof has a latent malaria infection.